Development of a Robust Bacillus amyloliquefaciens Cell Factory for Efficient Poly(γ-glutamic acid) Production from Jerusalem Artichoke

Bacillus amyloliquefaciens NB, a glutamate-independent poly-gamma-glutamic acid (gamma-PGA)-producing strain, can directly utilize inulin-containing sustainable materials. However, low gamma-PGA yield and lack of efficient genetic engineering approaches have hindered the industrial use of this strain. Here, we used the CRISPR-Cas9n technique to engineer B. amyloliquefaciens to enhance gamma-PGA production. We engineered three modules involved in inulin hydrolysis, reducing sugars metabolism, and gamma-PGA synthesis in B. amyloliquefaciens. Specifically, overexpresed the native inulin hydrolase CscA and two expression-optimized levanase and en doi nulinase, overexpressed of key genes related to reducing sugar metabolism to increased ATP production, and removed polysaccharide operon epsA-O and gamma-PGA hydrolase cwlO. Finally, the highest production of gamma-PGA (32.14 +/- 0.38 g/L) was obtained in a 7.5 L fed-batch fermenter. Thus, we successfully constructed an ideal candidate strain for efficient gamma-PGA production from inulin, which provides an important research basis for the development of more biobased products.